2.2.1. RNA-Dependent RNA Polymerase (RdRp) Inhibitors
The first licensed antiviral agents for COVID-19 were remdesivir and molnupiravir. They were approved for emergency use by the FDA to treat outpatients with mild COVID-19 phenotype and risk factors for progression to severe stages of the disease. Remdesivir is a non-canonical nucleotide able to stop the chain-elongation reaction of the viral RNA by RNA-dependent RNA polymerase (RdRp). Literature data gathered so far support the efficacy of intravenous remdesivir in reducing the risk and the length of hospitalization showing efficacy against several SARS-CoV-2 strains
[34]. The “WHO Solidarity randomized trial” investigated the efficacy of remdesivir in inpatients with COVID-19. Notably, 8275 patients were randomized to remdesivir or its control; mortality and hospitalization were chosen as primary and secondary outcomes, respectively. Overall mortality was 602 (14.5%) in patients receiving remdesivir with respect to 643 (15.6%) in the control group. A sub-analysis focused on the disease severity revealed that among the severe COVID-19 patients, 151 (42.1%) of 359 in treatment with remdesivir died compared to 134 (38.6%) of 347 in the control group. Of the patients receiving oxygen support, 14.6% assigned to remdesivir died versus 16.3% assigned to control
[35]. All these data showed a major efficacy of remdesivir in mild-to-moderate patients supporting the use of antivirals immediately following the onset of symptoms. Therefore, the current guidelines recommend remdesivir for unvaccinated ambulatory patients and vaccinated outpatients at risk for vaccine failure or at high risk for progression to severe disease within 7 days of symptom onset. The prodrug molnupiravir also inhibits RNA-polymerase of SARS-CoV-2 and other RNA viruses but presents a high mutational power in RNA strand, unlike remdesivir. Indeed, molnupiravir metabolization produces the cytidine nucleoside analogue N-hydroxycytidine (NHC) which is phosphorylated to the active form N-hydroxycytidine-5′-triphosphate (NHC-TP) into the cells. NHC monophosphate acts as a competitive substrate that will be incorporated by the SARS-CoV-2 viral RdRp introducing mutations (G to A and C to U substitution) in the viral genome and consequently inhibiting viral replication
[36][37]. The literature reports a higher antiviral activity of molnupiravir than remdesivir, likely due to the high binding stability of NHC monophosphate to RNA viral which prevents its removal by exonucleases. EUA was based on data from 1734 randomized mild-to-moderate COVID-19 participants, recruited in phase III MOVe-OUT trial. The study investigated the efficacy and safety of molnupiravir (800-mg) compared to placebo, delivered within 5 days of the onset of symptoms. Molnupiravir showed superiority over placebo in preventing hospitalization or death (7.3% versus 14.1%, respectively)
[38]. Nowadays, molnupiravir is licensed for adult unvaccinated outpatients and mild-to-moderate COVID-19 vaccinated patients at risk for vaccine failure, within 5 days from symptoms onset. However, molnupiravir use has not been recommended in international guidelines or is only recommended when no other treatment options exist due to its lower reported effectiveness at preventing hospitalization compared to other outpatient therapies.
2.2.2. Protease Inhibitors
M protease (Mpro) and PL protease (PLpro), respectively known as nsp5 and nsp3, are essential enzymes in the virus replication cycle, representing a therapeutic target to prevent the success of the infection. They process the virus polyproteins into active proteins and differ from human proteases, promoting the lower toxicity of protease inhibitors
[39]. Notably, nirmatrelvir (NM) is an oral protease inhibitor that is active by cleaving the 2 viral polyproteins; it is available in association with ritonavir
®, a strong cytochrome P450-3A4 inhibitor, that increases nirmatrelvir concentrations reducing the dose regimen and the side effects of the antiviral. Recent reports found that nirmatrelvir-ritonavir (NM/r) reduced the risk of death and hospitalization, showing more efficacy than molnupiravir. Moreover, data derived from the EPIC-HR trial (Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients) evaluating the efficacy of nirmatrelvir in non-hospitalized subjects without previous immunity against SARS-CoV-2 provided significant results on which the FDA EUA was based. The trial was performed when the delta variant was the predominant variant
[40][41]. A real-world interesting study was next carried out, based on data obtained from electronic medical records of the Israeli population. The aim of this work was to assess the effectiveness of NM/r in reducing the rate of hospitalizations in COVID-19 subjects when the omicron variant was the most common variant. The results corroborated the efficacy of NM/r in preventing severe COVID-19 onset, above all among adults 65 years of age or older
[42]. Overall, all these studies brought to light the importance to start NM/r treatment in early COVID-19 to prevent the worsening of the illness towards severe disease and quickly reduce SARS-CoV-2 viral load. Unfortunately, viral mutations represent the hot-spot for achieving successful therapy, probably responsible for the virological and clinical rebound in patients receiving NM/r as described by Charness et al.
[43]. Notably, the mechanisms involved in COVID-19 recrudescence after NM/r treatment were investigated, suggesting that reduced target drug concentrations related to pharmacokinetic changes and/or insufficient therapy length were involved
[44]. Moreover, some mutations in the Mpro have been revealed such as alanine 260 to threonine (A260T) or valine (A260V) in samples of patients receiving NM/r but did not cause a reduction in Mpro activity
[45]. Overall, the occurrence frequency of rebound illness is unclear, but increases in viral load were detected in 1 to 2% of participants in the phase III clinical trial
[46]. However, the mechanisms of COVID-19 recrudescence after treatment are still to be investigated. Currently, NMV/r is licensed for adult unvaccinated outpatients and vaccinated outpatients at risk for vaccine failure and/or progression to severe disease within 5 days of symptom onset.
3. Natural Products and Metal-Based Drugs as Adjuvants Agents in COVID-19 Control
To curb newly emerging SARS CoV2 variants, many plant metabolites may be valid alternatives against SARS-CoV-2. Among natural metabolites, alkaloids have potential drug activity by intercalation power against nucleic acids (DNA or RNA), stabilizing them in single-stranded form. Homoharringtonine (HHT) is a cytotoxic plant alkaloid
[47] strongly targeting the mRNA translation; its interesting antiviral activity promoted a protocol for clinical trials of HHT nebulization on COVID-19 patients has been registered (ChiCTR-2100045993) by the Ditan Hospital
[48]. Moreover, clinical trials and observational studies showed the positive effects of alkaloids colchicine and emetine in COVID-19 subjects
[49][50][51][52]. Carvacrol (CARV) is an essential oil extract derived from different plants which exerted antioxidant, antiviral and Ca
2+ influx modulator activities. Notably, Javed et al. reported a potential action in COVID-19, showing its potential inhibition activity of ACE2 and M
pro with a significant block in the host cell entry and replication of SARS-CoV-2 (
Figure 2)
[53][54]. Different research groups discovered the in vitro and in vivo anti-SARS-CoV-2 activity of cepharanthine, a natural alkaloid extracted from Stephania japonica. Cepharanthine have antioxidant and anti-inflammatory properties
[55][56] and could exert an antiviral effect by modulating Hypoxia-inducible factor-1 (HIF-1), a dysregulated factor in COVID-19
[57]. Recently, a high antiviral activity of cepharanthine against SARS-CoV-2 was identified, also preserving antiviral action against the Beta (B.1.351) variant. Thus, a phase II clinical study (NCT05398705) of cepharantine was completed in mild COVID-19 patients but the results are not posted yet. Finally, the LINCOLN survey showed the beneficial effects of Vitamin C and L-Arginine in long-COVID
[58][59][60] with significantly less severe long-COVID symptoms and a better effort perception when compared to the alternative treatment group
[61]. Overall, natural products represent an important tool in fighting COVID-19 but their use is only recommended in mild-to-moderate stages of the disease. Similarly, natural products as well as metal-based molecules may be valid tools for combating COVID-19. Phase II clinical trials are ongoing to evaluate the efficacy of ebselen (SPI-1005) in mild and severe COVID-19 patients (NCT04484025/NCT04483973). Ebselen is a synthetic organoselenium with a glutathione peroxidase function which exhibits in vitro antiviral activity against SARS-CoV-2
[62][63][64]. Iron level alterations are also detected in COVID-19 patients, related to oxidative stress. Therefore, iron chelators such as deferasirox, deferoxamine and deferiprone could be beneficial in decreasing cytotoxicity and oxidative stress damage associated with hyperferritinemia
[65]. Among them, deferiprone seems to be the most potent antioxidant drug in vitro, in vivo and in clinical models of COVID-like diseases, supporting its potential therapeutical use
[66][67].